Multifunctional selenium nanoparticle for treating inflammatory bowel disease as well as preparation method and application of multifunctional selenium nanoparticle

Abstract

The invention discloses multifunctional selenium nanoparticles for treating inflammatory bowel diseases as well as a preparation method and application of the multifunctional selenium nanoparticles, and relates to the technical field of biological medicines. The multifunctional selenium nanoparticle is of a core-shell structure and comprises an inner core and an outer layer coating the inner core, the inner core is selenium nanoparticles; and the outer layer is a polydopamine shell embedded with chitosan oligosaccharide. The multifunctional selenium nanoparticles provided by the invention have good stability and dispersibility, can target intestinal inflammation parts, prolong the in-vivo time of the nanoparticles, and show strong antioxidant and anti-inflammatory activity on cell and animal levels. The whole nanoparticle is based on low toxicity and colon targeting of a natural material, is higher in safety compared with a traditional glucocorticoid / biological preparation, relieves intestinal inflammation by adjusting a redox steady state, prolonging drug action time and the like, and is expected to become a novel colitis treatment strategy with high efficiency, targeting property and safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a multifunctional selenium nanoparticle for the treatment of inflammatory bowel disease, its preparation method, and its application. Background Technology

[0002] Colitis can be classified into specific and non-specific enteropathy based on its etiology. Inflammatory bowel disease (IBD), which has attracted considerable attention, belongs to the latter, including ulcerative colitis and Crohn's disease. Colitis patients experience symptoms such as vomiting, bloody diarrhea, abdominal pain, and weight loss, severely impacting their quality of life. In recent years, the incidence of IBD has been on the rise, becoming a significant health concern. Clinically, treatment involves oral, rectal, and systemic administration. Oral administration offers better compliance, convenience, and cost-effectiveness; however, the harsh gastrointestinal environment and rapid metabolism can reduce efficacy. Therefore, there is a need to develop drug delivery systems that can prolong the duration of action and improve bioavailability. Because small nanoparticles (<200nm) can easily penetrate the mucus layer and enhance drug efficacy by increasing drug concentration in diseased tissues, nanomedicine delivery systems show significant advantages in IBD treatment.

[0003] Excessive reactive oxygen species (ROS) disrupt the redox balance, triggering oxidative stress and inducing intestinal epithelial cell apoptosis and intestinal mucosal damage. This process not only accelerates mucosal ulcer formation but also triggers inflammatory responses, leading to the overactivation and recruitment of numerous immune cells to inflammatory sites. Therefore, eliminating excess ROS is a key strategy for alleviating intestinal inflammation and damage. Selenium (Se), an essential trace element for the human body, has been shown to effectively scavenge ROS in nanoparticles. However, chemically synthesized Se nanoparticles suffer from poor in vivo stability, often losing most of their activity before reaching inflamed tissues, which limits their bioavailability. Therefore, it is necessary to develop novel selenium nanoparticle drug delivery systems to improve the therapeutic effect on IBD. Summary of the Invention

[0004] The purpose of this invention is to provide a multifunctional selenium nanoparticle for treating inflammatory bowel disease, its preparation method, and its application, thereby addressing the problems existing in the prior art. This multifunctional selenium nanoparticle exhibits good stability and dispersibility, can target intestinal inflammation sites, prolongs the in vivo time of the nanoparticle, and displays strong antioxidant and anti-inflammatory activities at the cellular and animal levels.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a multifunctional selenium nanoparticle for treating inflammatory bowel disease, wherein the multifunctional selenium nanoparticle has a core-shell structure, comprising a core and an outer layer covering the core;

[0007] The core is a selenium nanoparticle; the outer layer is a polydopamine shell embedded with chitosan oligosaccharides.

[0008] The present invention also provides a method for preparing the above-mentioned multifunctional selenium nanoparticles, comprising the following steps:

[0009] An aqueous solution of selenium nanoparticles and a dopamine solution were added to an alkaline solution. After mixing and shaking, an aqueous solution of chitosan oligosaccharide was added, and the mixture was shaken again before centrifugation to obtain the multifunctional selenium nanoparticles.

[0010] Furthermore, the alkaline solution is a Tris-HCl solution with a pH of 8-9 or water.

[0011] Furthermore, the mixing and oscillating reaction time is 10 minutes.

[0012] Furthermore, the method for preparing selenium nanoparticles in the aqueous solution of selenium nanoparticles includes the following steps:

[0013] The reducing solution was added dropwise to a Na2SeO3 solution containing a dispersant, and the selenium nanoparticles were obtained through a redox reaction.

[0014] Furthermore, the reducing solution is an ascorbic acid solution.

[0015] Furthermore, the dispersant is Tween-20 or Tween-80.

[0016] The present invention also provides the application of the above-mentioned multifunctional selenium nanoparticles in the preparation of drugs for treating inflammatory bowel disease.

[0017] The present invention also provides a medicament for treating inflammatory bowel disease, the active ingredient of which includes the above-mentioned multifunctional selenium nanoparticles.

[0018] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0019] The present invention discloses the following technical effects:

[0020] (1) The oral multifunctional selenium nanoparticles prepared in this invention for the treatment of inflammatory bowel disease use Se-NPs synthesized by redox method as the core, and utilize the self-polymerization property of dopamine under alkaline conditions to coat the Se-NPs to form an outer layer. At the same time, chitosan oligosaccharide (COS) that promotes permeability and absorption is incorporated to form a stable nanodelivery system. Among them, selenium nanoparticles can effectively scavenge reactive oxygen species (ROS), and polydopamine (PDA) can exert its antioxidant activity and promote the targeted accumulation of nanoparticles at the site of inflammation due to its excellent adhesion. In addition, COS, as a water-soluble oligosaccharide, not only has multiple biological activities such as anti-inflammatory, antioxidant and immunomodulatory effects, but also enhances the permeability and absorption capacity of nanoparticles.

[0021] (2) The oral multifunctional selenium nanoparticles prepared in this invention for the treatment of inflammatory bowel disease combine Se, PDA, and COS. Compared with traditional selenium supplements, they enhance bioavailability and reduce the risk of liver and kidney damage, and are expected to solve the selenium deficiency problem in clinical colitis patients. After modification with PDA and COS, the anti-inflammatory and antioxidant properties are amplified several times, while significantly prolonging the retention time of nanoparticles at the site of intestinal inflammation. The nanoparticles as a whole are based on the low toxicity and colon-targeting properties of natural materials, and are safer than traditional glucocorticoids / biologics. By regulating redox homeostasis and prolonging the drug action time, they alleviate intestinal inflammation and are expected to become a new strategy for the treatment of colitis that combines high efficiency, targeting, and safety. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A representative atomic force microscope image of multifunctional selenium nanoparticles (SePC-NPs);

[0024] Figure 2 Particle size analysis diagram (A), zeta potential analysis diagram (B), and stability analysis diagram (C) of selenium nanoparticles (Se-NPs), selenium shield nanoparticles (SeP-NPs), and SePC-NPs;

[0025] Figure 3 The graph shows the results of antioxidant capacity determination of Se-NPs, SeP-NPs, and SePC-NPs; where A represents the ABTS scavenging results of different concentrations of SePC-NPs. + The experimental results of ·; B represents the scavenging of ABTS by different nanoparticles (Se equivalent concentration of 5 μg / mL). + C represents the experimental results of DPPH· scavenging by different concentrations of SePC-NPs; D represents the experimental results of DPPH· scavenging by different nanoparticles (Se equivalent concentration of 5 μg / mL).

[0026] Figure 4The graph shows the results of RAW264.7 cells co-incubating with fluorescently labeled selenium nanoparticles (RhB-Se-NPs), fluorescently labeled selenium shield nanoparticles (RhB-SeP-NPs), and fluorescently labeled multifunctional selenium nanoparticles (RhB-SePC-NPs) for different time periods, and examining the uptake of nanoparticles by cells using flow cytometry. In the graph, A is a representative histogram of RAW264.7 cells uptake of different nanoparticles at 4 h, and B is a bar chart of RAW264.7 cells uptake of different nanoparticles at different time periods.

[0027] Figure 5 Bar chart showing cell viability after co-incubation of RAW264.7 cells with Se-NPs, SeP-NPs, and SePC-NPs at different concentrations;

[0028] Figure 6 The cell viability bar chart (A) and cell live / dead staining chart (B) were obtained by co-incubating Se-NPs, SeP-NPs and SePC-NPs with hydrogen peroxide-induced RAW264.7 cells.

[0029] Figure 7 Representative flow cytometry (A) and bar graph (B) of nanoparticle scavenging reactive oxygen species (ROS) levels were determined by co-incubating Se-NPs, SeP-NPs and SePC-NPs with lipopolysaccharide-induced RAW264.7 cells using the DCFH-DA probe.

[0030] Figure 8 The images show the in vivo detection results of colonic inflammation sites in mice after oral administration of fluorescently labeled selenium shield nanoparticles (Cy7-SeP-NPs) and fluorescently labeled multifunctional selenium nanoparticles (Cy7-SePC-NPs). In the figures, A is the in vivo fluorescence distribution map; B is the in vivo fluorescence intensity quantitative line graph; C is the in vitro fluorescence distribution map; and D is the in vitro fluorescence intensity quantitative bar graph. Control SePC-NPs represents the control group; DSS SeP-NPs represents the Cy7-SeP-NPs group; and DSS SePC-NPs represents the Cy7-SePC-NPs group.

[0031] Figure 9 Line graph (A) showing changes in body weight, line graph (B) showing changes in disease activity index, comparison graph (C) showing colon length, and bar graph (D) showing colon length in mice after oral treatment with Se-NPs, SeP-NPs, and SePC-NPs;

[0032] Figure 10 H&E staining images of major organs in mice after oral administration of Se-NPs, SeP-NPs and SePC-NPs. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] All raw materials used in the following examples can be purchased through commercial channels.

[0039] Example 1

[0040] A method for preparing multifunctional selenium nanoparticles (SePC-NPs) includes the following steps:

[0041] (1) Weigh 26.3 mg of sodium selenite pentahydrate and prepare a 2.63 mg / mL sodium selenite aqueous solution. Simultaneously, weigh 70 mg of ascorbic acid and prepare a 7 mg / mL ascorbic acid aqueous solution. Place 10 mL of the sodium selenite aqueous solution in a reaction flask, add 200 μL of Tween-20 solution as a dispersant, and finally add 10 mL of ascorbic acid aqueous solution dropwise. Mix and react at 500 rpm for 12 h at 25 °C. Centrifuge the solution (10000 g, 30 min) and wash to obtain selenium nanoparticles (Se-NPs). Resuspend the Se-NPs in 6 mL of pure water to obtain a Se-NPs aqueous solution for later use.

[0042] (2) Dilute 1M Tris-HCl to 10mM, adjust the pH to 9.0 with NaOH solution, weigh out dopamine hydrochloride solution and dissolve it in Tris-HCl to prepare a 10mg / mL dopamine hydrochloride solution, and dissolve chitosan oligosaccharide (COS) in water to prepare a 2mg / mL COS aqueous solution. Take 3.65mL of Tris-HCl solution, add 150μL of Se-NPs aqueous solution (Se concentration is 183.8μg / mL) and 200μL of dopamine hydrochloride solution, shake at 3000rpm, starting from the 10th min, add 20μL of COS aqueous solution every 30min, shake for a total of 2h, then centrifuge (10000g, 30min) and wash to obtain SePC-NPs.

[0043] Example 2

[0044] A method for preparing multifunctional selenium nanoparticles includes the following steps:

[0045] (1) Weigh 26.3 mg of sodium selenite pentahydrate and prepare a 2.63 mg / mL sodium selenite aqueous solution. Simultaneously, weigh 70 mg of ascorbic acid and prepare a 7 mg / mL ascorbic acid aqueous solution. Place 10 mL of the sodium selenite aqueous solution in a reaction flask, add 200 μL of Tween-20 solution as a dispersant, and finally add 10 mL of ascorbic acid aqueous solution dropwise. Mix and react at 550 rpm for 14 h at 20 °C. Centrifuge the solution (9500 g, 60 min) and wash to obtain Se-NPs. Resuspend the Se-NPs in 6 mL of pure water to obtain a Se-NPs aqueous solution for later use.

[0046] (2) Adjust the pH of the water to 8.0 with NaOH solution. Weigh out the dopamine hydrochloride solution and dissolve it in water at pH 8.0 to prepare a 0.5 mg / mL dopamine hydrochloride solution. Dissolve COS in water to prepare a 5 mg / mL COS aqueous solution. Take 3.65 mL of water at pH 8.0, add 150 μL of Se-NPs aqueous solution (Se concentration of 183.8 μg / mL) and 200 μL of dopamine hydrochloride solution, and shake at 3000 rpm. Starting from the 10th minute, add 200 μL of COS aqueous solution every 30 minutes, and shake for a total of 2 hours. Then centrifuge (15000g, 30 min) and wash to obtain SePC-NPs.

[0047] Example 3

[0048] A method for preparing multifunctional selenium nanoparticles (SePC-NPs) includes the following steps:

[0049] (1) Weigh 26.3 mg of sodium selenite pentahydrate and prepare a 2.63 mg / mL sodium selenite aqueous solution. Simultaneously, weigh 70 mg of ascorbic acid and prepare a 7 mg / mL ascorbic acid aqueous solution. Place 10 mL of the sodium selenite aqueous solution in a reaction flask, add 200 μL of Tween-80 solution as a dispersant, and finally add 10 mL of ascorbic acid aqueous solution dropwise. Mix and react at 600 rpm for 8 h at room temperature. Centrifuge the solution (15000 g, 10 min) and wash to obtain Se-NPs. Resuspend the Se-NPs in 6 mL of pure water to obtain a Se-NPs aqueous solution for later use.

[0050] (2) Dilute 1M Tris-HCl to 10mM, adjust the pH to 8.5 with NaOH solution, weigh out dopamine hydrochloride solution and dissolve it in Tris-HCl to prepare a 5mg / mL dopamine hydrochloride solution, and dissolve COS in water to prepare a 1mg / mL COS aqueous solution. Take 3.65mL of Tris-HCl solution, add 150μL of Se-NPs aqueous solution (Se concentration is 183.8μg / mL) and 200μL of dopamine hydrochloride solution, shake at 3000rpm, starting from the 10th min, add 10μL of COS aqueous solution every 30min, shake for a total of 2h, then centrifuge (10000g, 30min) and wash to obtain SePC-NPs.

[0051] Comparative Example 1

[0052] A method for preparing selenium nanoparticle cores (Se-NPs) includes the following steps:

[0053] Weigh 26.3 mg of sodium selenite pentahydrate and prepare a 2.63 mg / mL sodium selenite aqueous solution. Simultaneously, weigh 70 mg of ascorbic acid and prepare a 7 mg / mL ascorbic acid aqueous solution. Place 10 mL of the sodium selenite aqueous solution in a reaction flask, add 200 μL of Tween-20 solution as a dispersant, and finally add 10 mL of ascorbic acid aqueous solution dropwise. Mix and react at 25 °C and 500 rpm for 12 h. Centrifuge the solution (10000 g, 30 min) and wash to obtain Se-NPs.

[0054] Comparative Example 2

[0055] A method for preparing selenium shield nanoparticles (SeP-NPs) includes the following steps:

[0056] (1) Weigh 26.3 mg of sodium selenite pentahydrate and prepare a 2.63 mg / mL sodium selenite aqueous solution. Simultaneously, weigh 70 mg of ascorbic acid and prepare a 7 mg / mL ascorbic acid aqueous solution. Place 10 mL of the sodium selenite aqueous solution in a reaction flask, add 200 μL of Tween-20 solution as a dispersant, and finally add 10 mL of ascorbic acid aqueous solution dropwise. Mix and react at 500 rpm for 12 h at 25 °C. Centrifuge the solution (10000 g, 30 min) and wash to obtain selenium nanoparticles (Se-NPs). Resuspend the Se-NPs in 6 mL of pure water to obtain a Se-NPs aqueous solution for later use.

[0057] (2) Dilute 1M Tris-HCl to 10mM, adjust the pH to 9.0 with NaOH solution, and dissolve dopamine hydrochloride solution in Tris-HCl to prepare a 10mg / mL dopamine hydrochloride solution. Take 3.65mL of Tris-HCl solution, add 150μL of Se-NPs aqueous solution (Se concentration of 183.8μg / mL) and 200μL of dopamine hydrochloride solution, and shake for 2h. Centrifuge the solution (10000g, 30min) and wash to obtain SeP-NPs.

[0058] Example 4

[0059] A method for preparing rhodamine B fluorescently labeled Se-NPs, SeP-NPs, and SePC-NPs includes the following steps:

[0060] 150 μL of Se-NPs from Comparative Example 1 was dissolved in 850 μL of pure water. Meanwhile, the SeP-NPs obtained from Comparative Example 2 and the SePC-NPs obtained from Example 1 were resuspended in 1 mL of pure water, respectively. Then, all three solutions were mixed with 1 mL of Rhodamine B aqueous solution (1 mg / mL) and shaken at 3000 rpm for 4 h. The shaken solutions were centrifuged (10000 g, 30 min) and washed to obtain different Rhodamine B-labeled nanoparticles: RhB-Se-NPs, RhB-SeP-NPs, and RhB-SePC-NPs.

[0061] Example 5

[0062] A method for preparing Cy7-NHS fluorescently labeled SeP-NPs and SePC-NPs includes the following steps:

[0063] The Se-NPs prepared in Comparative Example 1 were resuspended in 6 mL of pure water to obtain an aqueous solution of Se-NPs. 1 M Tris-HCl was diluted to 10 mM, and the pH was adjusted to 9.0 with NaOH solution. Dopamine hydrochloride solution was dissolved in Tris-HCl to prepare a 10 mg / mL dopamine hydrochloride solution, and COS was dissolved in water to prepare a 2 mg / mL COS aqueous solution. 3.65 mL of Tris-HCl solution was added, along with 150 μL of the Se-NPs aqueous solution and 200 μL of the dopamine hydrochloride solution, and the mixture was shaken at 3000 rpm. After 10 min, 1 mg Cy7-NHS and 20 μL of COS aqueous solution were added, with 20 μL of COS aqueous solution added every 30 min, and the mixture was shaken for a total of 2 h. The solution was centrifuged (10000 g, 30 min) and washed to obtain Cy7-SePC-NPs.

[0064] The preparation process of Cy7-SeP-NPs is the same as that of Cy7-SePC-NPs, except that COS aqueous solution is not added during the preparation process.

[0065] To better characterize the properties of multifunctional selenium nanoparticles and their efficacy in treating inflammatory bowel disease, the following experiments were conducted:

[0066] Experimental Example 1

[0067] Morphological characterization was performed on the SePC-NPs prepared in Example 1 of this invention:

[0068] SePC-NPs were adjusted to a suitable concentration, dropped onto mica sheets, and allowed to dry naturally. The morphology of the nanoparticles was then characterized using atomic force microscopy. The results are as follows: Figure 1 As shown, SePC-NPs exhibit a spherical structure with small particle size and uniform distribution.

[0069] Experimental Example 2

[0070] The particle size, potential, and stability of the Se-NPs prepared in Comparative Example 1, the SeP-NPs prepared in Comparative Example 2, and the SePC-NPs prepared in Example 1 were determined.

[0071] The particle size and potential of the nanoparticles were analyzed using a Malvern particle size potentiometer; the nanoparticles were stored at 4°C, and their stability was demonstrated by measuring their particle size for seven consecutive days. Figure 2 As shown in Figure A, the average particle sizes of Se-NPs, SeP-NPs, and SePC-NPs are 53.06±0.32 nm, 120.6±2.3 nm, and 141.9±3.6 nm, respectively, all exhibiting a normal distribution and a polydispersity index < 0.1, indicating that the nanoparticles possess good uniformity and dispersibility. Furthermore, the particle size measurement results of SePC-NPs are consistent with the atomic force diagram in Experimental Example 1. Figure 2 As shown in Figure B, the Zeta potentials of Se-NPs, SeP-NPs, and SePC-NPs are -20.95±1.18 mV, -17.33±0.45 mV, and -6.47±0.69 mV, respectively. The addition of COS increases the potential of the nanoparticles, indicating successful COS incorporation. Figure 2 As shown in Figure C, the particle size of the three types of nanoparticles did not change significantly within 7 days at 4℃, indicating that the nanoparticles have a certain degree of stability.

[0072] Experimental Example 3

[0073] The free radical scavenging abilities of Se-NPs prepared in Comparative Example 1, SeP-NPs prepared in Comparative Example 2, and SePC-NPs prepared in Example 1 were determined:

[0074] Prepare the ABTS test kit in advance according to the instructions for the Total Antioxidant Capacity Assay Kit (ABTS method). + Mother liquor and diluted to ABTS + • Working solution. Take 100 μL of aqueous solutions of nanoparticles of different concentrations and mix with 200 μL of LABTS. + • After mixing the working solutions and incubating at room temperature for 10 min, the absorbance of each solution at 734 nm was measured using a microplate reader. • A 0.1 mM 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution was prepared using ethanol. 100 μL of different concentrations of nanoparticle aqueous solutions were mixed with 100 μL of DPPH solution, and after incubation at room temperature for 30 min, the absorbance of each solution at 517 nm was measured using a microplate reader. For both methods, equal volumes of pure water and ABTS were used for each measurement. + • A mixture of working solution or DPPH solution was used as a blank control, and an equal volume of nanoparticle aqueous solution was mixed with pure water as a self-control.

[0075] The formula for calculating the free radical scavenging rate is:

[0076] Free radical scavenging rate (%) = 1 - (A 样品 -A 自身 ) / A 空白 ×100%.

[0077] like Figure 3 As shown in Figures A and B, when the concentration is 2.5 μg / mL, SePC-NPs have a significant effect on ABTS. + The scavenging rate of · was 62%, and the ABTS of SePC-NPs was 5 μg / mL. + • The clearance rate reaches 97%, which is significantly different from other nanoparticles. For example... Figure 3 As shown in C and D, when the concentration was 2.5 μg / mL, SePC-NPs scavenged 75% of DPPH·, indicating that SePC-NPs have excellent antioxidant properties.

[0078] Test Example 4

[0079] The uptake of RhB-Se-NPs, RhB-SeP-NPs, and RhB-SePC-NPs prepared in Example 4 by mouse macrophage RAW264.7 cells was measured:

[0080] RAW264.7 mouse macrophages with good growth status were used at 5 × 10⁻⁶. 4 RhB-Se-NPs, RhB-SeP-NPs, and RhB-SePC-NPs were seeded at a density of 1 nanoparticle / well in 24-well plates. After 12 h of culture, the culture medium was discarded, and RhB-Se-NPs, RhB-SeP-NPs, and RhB-SePC-NPs resuspended in basal medium were added. The equivalent concentration of Se in each nanoparticle was 5 μg / mL. Cells were collected and washed at 0 h, 2 h, 4 h, 6 h, and 12 h of incubation with the nanoparticles. After resuspending in 300 μL of 4% tissue fixative and sieving, the uptake efficiency of each nanoparticle by mouse macrophage RAW264.7 cells was detected by flow cytometry.

[0081] The results are as follows Figure 4 As shown, the uptake efficiency of RAW264.7 cells for each nanoparticle increased with time. However, at 4 h, the uptake efficiency of RAW264.7 cells for RhB-SePC-NPs reached 84.2%, which was much higher than that for RhB-Se-NPs and RhB-SeP-NPs. This may be because the modification of COS weakened the electrostatic repulsion between Se-NPs and the negatively charged cell membrane. At the same time, the phenolic hydroxyl and amino groups on the surface of polydopamine (PDA) may have strengthened the interaction with the cell membrane through hydrogen bonding and electrostatic interaction, thus promoting the uptake of nanoparticles by RAW264.7 cells.

[0082] Experimental Example 5

[0083] Tests were conducted to determine the toxicity of Se-NPs prepared in Comparative Example 1, SeP-NPs prepared in Comparative Example 2, and SePC-NPs prepared in Example 1 to mouse macrophage RAW264.7 cells.

[0084] RAW264.7 macrophages were used at a concentration of 1×10⁻⁶. 4 The nanoparticles were seeded at a density of 1 nanoparticle / well in 96-well plates. After 12 hours of culture, the culture medium was discarded, and different concentrations of Se-NPs, SeP-NPs, and SePC-NPs were resuspended in basal medium and co-incubated. The control group was incubated with basal medium instead of the drug-containing medium. After 24 hours, the culture medium was discarded and the cells were washed with PBS. The absorbance at 490 nm was measured using an MTS kit to indicate whether different concentrations of each nanoparticle had a toxic effect on RAW264.7 cells.

[0085] The results are as follows Figure 5 As shown, at Se equivalent concentrations below 10 μg / mL, neither SeP-NPs nor SePC-NPs exhibited significant toxicity to RAW264.7 cells. However, when the Se equivalent concentration was 10 μg / mL, the survival rate of RAW264.7 cells treated with Se-NPs was only 34.72%. This may be related to the certain biocompatibility of PDA and COS, and the fact that PDA and COS form a coating as a physical barrier, controlling the release rate of Se and avoiding cytotoxicity caused by high-concentration Se exposure in a short period of time.

[0086] Experimental Example 6

[0087] Determination of the ability of Se-NPs prepared in Comparative Example 1, SeP-NPs prepared in Comparative Example 2, and SePC-NPs prepared in Example 1 to protect mouse macrophages RAW264.7 against oxidative stress:

[0088] RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 4 RAW264.7 cells were seeded at a density of 10 cells / well in 96-well plates and cultured for 12 hours. The culture medium was then discarded. The control group received basal culture medium, while the remaining cells received 300 μM H2O2 solution to stimulate oxidative stress. Simultaneously, Se-NPs, SeP-NPs, and SePC-NPs (Se equivalent concentration of 5 μg / mL) resuspended in basal culture medium were added and incubated for 12 hours. The drug-containing culture medium was then discarded, and the cells were washed with PBS. The absorbance at 490 nm was measured using an MTS assay kit. Additionally, RAW264.7 cells were seeded at a density of 1 × 10⁻⁶ cells / well. 5Cells were seeded at a density of [number] cells / well in 12-well plates and cultured for 12 hours. The culture medium was then discarded. The control group received basal culture medium, while the remaining cells received 1 mM H₂O₂ solution to stimulate oxidative stress. Simultaneously, Se-NPs, SeP-NPs, and SePC-NPs (equivalent concentration of Se 5 μg / mL) resuspended in basal culture medium were added and incubated for 12 hours. The drug-containing culture medium was then discarded, and the cells were washed with PBS. Cell viability and cytotoxicity were assessed using the Calcein-AM / PI cell viability and cytotoxicity assay kit according to the manufacturer's instructions. The cells were observed and photographed under an inverted fluorescence microscope. Results are as follows: Figure 6 As shown.

[0089] Figure 6 In Figure A, cell viability was detected by the MTS method. Hydrogen peroxide-induced oxidative stress killed 20.32% of RAW264.7 cells, but cell viability recovered to 83.22%, 95.60%, and 101.62% after incubation with Se-NPs, SeP-NPs, and SePC-NPs, respectively. Figure 6 In section B, double staining of live and dead cells was observed using an inverted fluorescence microscope. The addition of SePC-NPs significantly reduced the number of PI-labeled dead cells, compared with... Figure 6 The results from both A and B corroborate each other, indicating that multifunctional selenium nanoparticles can protect cells from oxidative stress damage caused by exogenous stimuli. This is because PDA is rich in catechol groups, and the reducing amino groups contained in COS can neutralize reactive oxygen species and scavenge oxidative stress. At the same time, compared with Se-NPs and SeP-NPs, SePC-NPs can be taken up by cells more rapidly, thus exhibiting a stronger efficacy in scavenging oxidative stress.

[0090] Experimental Example 7

[0091] Determination of the ability of Se-NPs prepared in Comparative Example 1, SeP-NPs prepared in Comparative Example 2, and SePC-NPs prepared in Example 1 to scavenge reactive oxygen species (ROS) in mouse macrophages RAW264.7:

[0092] RAW264.7 macrophages were used at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured for 12 h. The culture medium was then discarded, and 1 μg / mL lipopolysaccharide (LPS) was added to induce an inflammatory environment for 6 h, generating ROS. After successful induction, LPS was discarded, and PBS, Se-NPs, SeP-NPs, and SePC-NPs (Se equivalent concentration of 5 μg / mL) were added and co-incubated for 12 h. The control group used an equal volume of basal culture medium instead of LPS and the drug-containing medium throughout the process. After washing with PBS, the DCFH-DA fluorescent probe (10 μM) was added and incubated at 37°C for 30 min. Cells were collected, washed three times with PBS, resuspended in 300 μL PBS, sieved, and then quantitatively detected using flow cytometry.

[0093] The results are as follows Figure 7 As shown, the ROS level in the control group without LPS induction was 0.22%, and the ROS level in the PBS group was 5.20%. However, after incubation with Se-NPs, SeP-NPs, and SePC-NPs, the ROS levels in each group decreased to 2.59%, 0.66%, and 0.34%, respectively, showing statistically significant differences. This may be due to the synergistic effect of PDA and COS in directly scavenging oxidative stress and the anti-inflammatory activity of nanoparticles in resisting the pro-inflammatory effect of LPS.

[0094] Experimental Example 8

[0095] Tests were conducted on the in vivo retention of fluorescently labeled selenium shield nanoparticles (Cy7-SeP-NPs) and fluorescently labeled multifunctional selenium nanoparticles (Cy7-SePC-NPs) at the site of colonic inflammation prepared in Example 5:

[0096] A mouse model of colitis was established by allowing free access to a 3% sodium dextran sulfate solution. Successful model establishment was defined as a weight loss greater than 15% or a disease activity index greater than or equal to 2. Model mice were randomly divided into Cy7-SeP-NPs and Cy7-SePC-NPs groups and administered the drugs via gavage. Healthy mice served as a control group and were given the same amount of Cy7-SePC-NPs via gavage. In vivo imaging was performed at 1 h, 2 h, and 4 h post-administration to observe the distribution of Cy7-SePC-NPs in vivo. Mice were then euthanized by cervical dislocation, and the gastrointestinal tract was harvested for in vitro imaging. Fluorescence intensity was quantified in both in vivo and in vitro imaging. Results are as follows: Figure 8 As shown.

[0097] Figure 8 Image A shows the distribution of the mice in vivo at 1 hour, 2 hours, and 4 hours after gavage. Figure 8 B represents the quantitative analysis of average fluorescence intensity. It shows that there was almost no difference in fluorescence intensity among the three groups 1 hour after administration. However, as time increased, the fluorescence intensity of the control group gradually decreased, while the Cy7-SePC-NPs group had the highest fluorescence intensity and the most targeted accumulation at 4 hours after administration. Figure 8 Image C shows isolated gastrointestinal tract images of mice in each group 4 hours after drug administration. Figure 8 The middle D represents the quantitative analysis of total fluorescence intensity in the colon, showing that the model mice treated with Cy7-SePC-NPs accumulated the most nanoparticles in the colon at 4 hours, compared with... Figure 8 China A Figure 8 The in vivo imaging results of B corroborate each other, indicating that the PDA coating has a certain degree of adhesion. At the same time, the permeation-enhancing effect of COS synergistically enhances the accumulation of nanoparticles in the intestinal inflammatory site. The increase in macrophages in the inflammatory site and the faster uptake of nanoparticles by macrophages may also be one of the reasons for the increased accumulation of nanoparticles in the inflammatory site.

[0098] Experimental Example 9

[0099] The efficacy of Se-NPs prepared in Comparative Example 1, SeP-NPs prepared in Comparative Example 2, and SePC-NPs prepared in Example 1 in treating mice with colitis was investigated.

[0100] A mouse colitis model was established by allowing free access to 3% sodium dextran sulfate solution for 5 consecutive days. Model establishment was considered successful when body weight decreased by more than 15% or the disease activity index was greater than or equal to 2. Model mice were randomly divided into 5 groups and administered PBS, Se-NPs, SeP-NPs, or SePC-NPs every 24 hours via gavage for 3 consecutive days. Healthy mice served as a control group, and these mice had free access to sterile water during the modeling period, receiving an equal volume of PBS via gavage at the time of drug administration. From the start of model establishment, mouse body weight (and during drug administration, body weight was recorded after each administration) and disease activity index were recorded daily. The disease activity index criteria were based on Table 1, and the final score was recorded as the average of the three criteria.

[0101] Table 1. Standards for Disease Activity Index

[0102]

[0103] Mice were euthanized by cervical dislocation on day 9. The cecum, colon, and rectum were harvested to measure the length of the colon and rectum, and images were taken and recorded. The results are as follows: Figure 9 As shown. Figure 9 Figure A shows the changes in mouse body weight. Figure 9 In the diagram, B represents the disease activity index. 24 hours after the first administration, the weight loss of mice in the SePC-NPs group slowed down, and the disease activity index score decreased significantly. After the third administration, the weight of mice in the SePC-NPs group rebounded, and the disease activity index approached 1. This efficacy was greater than that of SeP-NPs and Se-NPs. Figure 9 Image C shows the colon of mice after three doses. Figure 9 The quantitative analysis of colon length in the middle section (D) revealed that the colon length of mice treated with SePC-NPs was significantly restored. Furthermore, SeP-NPs and Se-NPs also promoted the restoration of colon length in the model mice to some extent. This indicates that Se-NPs, SeP-NPs, and SePC-NPs all have therapeutic efficacy in treating colitis in mice, with SePC-NPs exhibiting the best therapeutic efficacy.

[0104] Experimental Example 10

[0105] Evaluation of the in vivo biocompatibility of Se-NPs prepared in Comparative Example 1, SeP-NPs prepared in Comparative Example 2, and SePC-NPs prepared in Example 1:

[0106] A mouse colitis model was established by allowing free access to 3% sodium dextran sulfate solution for 5 consecutive days. Successful model establishment was defined as a weight loss greater than 15% or a disease activity index greater than or equal to 2. The model mice were randomly divided into four groups. Three groups received Se-NPs, SeP-NPs, or SePC-NPs, respectively, administered every 24 hours via gavage for 3 consecutive days. The remaining group served as a control group (healthy mice), providing free access to sterile water during the modeling period and receiving an equal volume of PBS via gavage during treatment. After treatment, mice were euthanized by cervical dislocation. Their hearts, livers, spleens, lungs, and kidneys were harvested, dehydrated in paraffin, embedded, sectioned, and stained using the H&E kit according to the manufacturer's instructions. The sections were then scanned using a digital slide scanner. Results are as follows: Figure 10 As shown, no obvious pathological abnormalities were observed in the major organs of mice in each treatment group, indicating that the nanoparticles have no obvious toxic side effects on normal tissues and have high biosafety.

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multifunctional selenium nanoparticle for treating inflammatory bowel disease, characterized in that, The multifunctional selenium nanoparticles have a core-shell structure, including a core and an outer layer covering the core; The core is a selenium nanoparticle; the outer layer is a polydopamine shell embedded with chitosan oligosaccharides.

2. A method for preparing multifunctional selenium nanoparticles as described in claim 1, characterized in that, Includes the following steps: An aqueous solution of selenium nanoparticles and a dopamine solution were added to an alkaline solution. After mixing and shaking, an aqueous solution of chitosan oligosaccharide was added, and the mixture was shaken again before centrifugation to obtain the multifunctional selenium nanoparticles.

3. The preparation method according to claim 2, characterized in that, The alkaline solution is a Tris-HCl solution with a pH of 8-9 or water.

4. The preparation method according to claim 2, characterized in that, The mixing and oscillating reaction time is 10 min.

5. The preparation method according to claim 2, characterized in that, The method for preparing selenium nanoparticles in the aqueous solution includes the following steps: The reducing solution was added dropwise to a Na2SeO3 solution containing a dispersant, and the selenium nanoparticles were obtained through a redox reaction.

6. The preparation method according to claim 5, characterized in that, The reducing solution is an ascorbic acid solution.

7. The preparation method according to claim 5, characterized in that, The dispersant is Tween-20 or Tween-80.

8. The use of the multifunctional selenium nanoparticles as described in claim 1 in the preparation of a medicament for treating inflammatory bowel disease.

9. A drug for treating inflammatory bowel disease, characterized in that, The active ingredient includes the multifunctional selenium nanoparticles as described in claim 1.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images